Medicinal uses of dihydropyrazoles

ABSTRACT

Compounds having a structure according to Formula (I):  
                 
are effective in a method of increasing erythropoietin and vascularization of tissue in a subject in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under Title 35, United States Code119(e) from Provisional Application Ser. No. 60/288,720, filed May 4,2001.

TECHNICAL FIELD

This invention is directed to compounds that are useful in methods oftreating hypoxia related disorders. The invention is also directed topharmaceutical compositions comprising the compounds.

BACKGROUND

Ischemic cardiac disease and peripheral vascular disease are majorhealth problems affecting hundreds of millions of people worldwide.Ischemia results when there is a lack of oxygen supply. It is estimatedthat about half of the deaths that occur in the United States each yearalone are caused by ischemic heart disease. This invention relates, inpart, to methods for the treatment of such diseases and pharmaceuticalcompositions in the treatment thereof.

Oxygen is essential for an organism's survival, given its role inessential metabolic processes including oxidative phosphorylation inwhich O₂ serves as electron acceptor during ATP formation. Tissue damagecan result from hypoxia that is, when oxygen supply in tissue isinsufficient to meet metabolic demands. Hypoxia can be caused by variousmedical conditions, including atherosclerosis, chronic illness, trauma,and surgical procedures. Accordingly, hypoxia plays an important role inthe pathogenesis of major causes of mortality, including cancer,cerebral and myocardial ischemia, and chronic heart and lung diseases.

Organisms can sense O₂ concentration and adaptively respond to hypoxia.These adaptive responses either increase O₂ delivery or activatealternative metabolic pathways that do not require O₂. There are anumber of hypoxia-inducible gene products that participate in theseresponses. Included, are genes that code for erythropoietin (hereinafter“EPO”), vascular endothelia growth factor (hereinafter “VEGF”), tyrosinehydroxylase, and glycolytic enzymes. See Bunn, H. F & Poyton, R. O.,“Oxygen sensing and molecular adaptation to hypoxia”, Physiol. Rev.,Vol. 76, pp. 839-885 (1996): Semenza, G. L., “Regulation of mammalian O₂homeostasis by hypoxia-inducible factor 1”, Annu. Rev. Cell. Dev. Biol.,Vol. 15, pp. 551-57

(1999); Shweiki, D. et al., “Induction of vascular endothelial growthfactor expression by hypoxia and by glucose deficiency in multicellspheroids: implications for tumor angiogenesis”, Proc. Natl. Acad. Sci.U.S.A., Vol. 92, pp. 768-772 (1995). The transcriptional regulatorhypoxia-inducible factor 1 (hereinafter “HIF1”) is an essential mediatorof O₂ homeostasis and regulates the transcription rate of many genesincluding the aforementioned genes. See Wang, G. L., et al., Biochem.Biophys. Res. Commun., Vol. 86, pp. 15-22 (1995). The number of targetgenes activated by HIF-1 includes genes whose protein products areinvolved in angiogenesis, energy metabolism, erythropoiesis, cellproliferation and viability, vascular remodeling, and vasomotorresponses. Semenza, G. L., “HIF-1: mediator of physiological andpathophysiological responses to hypoxia.” J. Appl. Physiol. Vol. 88, pp.1474-1480 (2000); Semenza, G. L., “Hypoxia-inducible factor 1: masterregulator of O₂homeostasis,” Genetics & Development, Vol. 8, pp. 588-594(1998).

Structurally, HIF-1 is a heterodimer of two subunits, HIF-1α and HIF-1β.The biological activity of HIF-1 is determined by the expression andactivity of the HIF-1α subunit. See Jiang, B-H. et al., “Transactivationand inhibitory domains of hypoxia-inducible factor 1α: modulation oftranscriptional activity by oxygen tension”, J. Biol. Chem., Vol. 272,pp. 19253-60 (1997). The in vivo regulation of HIF-1α biologicalactivity occurs at multiple levels, including mRNA expression, proteinexpression, nuclear localization, and transactivation. Semenza, J. Appl.Physiol., Vol. 88, page 1476 (2000). Hypoxia, in turn, is known to haveat least two independent effects on HIF-1α activity: (1) hypoxiaincreases the steady-state levels of HIF-1α protein by stabilizing it(i.e., decreasing its degradation); and (2) hypoxia increases thespecific transcriptional activity of the protein (i.e., independent ofthe protein concentration). Jiang, B. H., et al., “Dimerization, DNAbinding and transactivation properties of hypoxia-inducible factor 1,”J. Biol. Chem., Vol. 271, pp. 17771-78 (1996). Given HIF-1's role inhypoxia, treatments utilizing HIF-1 in the treatment of hypoxia-relateddisorders, have been described. For example, U.S. Pat. Nos. 5,882,914;6,020,462; 6,124,131 and international publication number WO 00/10578.

Although the known number of target genes activated by HIF-1 continuesto increase, the role of HIF-1 in the activation of VEGF genetranscription in hypoxic cells is well established. Semenza, J. Appl.Physiol, Vol. 88, page 1477 (2000). VEGF itself mediates a number ofresponses including vasodilation, vascular permeability, and endothelialcell migration and proliferation through receptors that are restrictedto vascular endothelium and certain hematopoietic cells. The combinedeffects of VEGF are important to the promotion of an angiogenicresponse. The restricted localization of VEGF receptors provides a levelof specificity that makes VEGF an important target for angiogenictherapy. For example, the promotion of blood vessel growth has beendemonstrated in animal models of coronary and limb ischemia. SeePearlman, J. D., et al., “Magnetic resonance mapping demonstratesbenefits of VEGF-induced myocardial angiogenesis”, Nat. Med. Vol. 1. pp.1085-1089 (1995); Takeshita, S., et al., “Therapeutic angiogenesis. Asingle intraarterial bolus of vascular endothelial growth factoraugments revascularization in a rabbit ischemic hind limb model”, J.Clin. Invest., Vol. 93, pp. 662-670 (1994). There are several clinicaltrials in progress to assess the efficacy of both exogenouslyadministered VEGF protein as well as expression vectors for the VEGFgene. See Hendel, R. C., et al., “Effect of intracoronary recombinanthuman vascular endothelial growth factor on myocardialperfusion—Evidence for a dose-dependent effect”, Circulation, Vol.101(2), pp. 118-121 (2000); Schwarz, et al., “Evaluation of the effectsof intramyocardial injection of DNA expressing vascular endothelialgrowth factor in a myocardial infarction model in the rat—Angiogenesisand angioma formation”, J. Amer. Coll. Cardiol., Vol. 35(5), pp.1323-1330 (2000).

Another approach to utilizing the effects of VEGF in proangiogenictherapy is to stimulate its production from the tissues needing newvessels. Secretion of VEGF appears to be dependent on its rate ofbiosynthesis since the intracellular storage of VEGF protein has notbeen demonstrated. The biosynthesis of VEGF is primarily controlled byregulating the amount of VEGF mRNA. See Shweiki, D., et al, “Vascularendothelial growth factor induced by hypoxia may mediatehypoxia-initiated angiogenesis”, Nature, Vol. 359. pp. 843-845 (1992).In turn, the amount of mRNA is controlled by activation of transcriptionthrough regulatory elements located in the 5′ promoter sequence of theVEGF gene as well as by less characterized mechanisms that stabilizeVEGF mRNA. See Levy, A. P., et al., “Transcriptional regulation of therat vascular endothelial growth factor gene by hypoxia”, Drug Discov.Today, Vol. 270, pp. 13333-13340 (1995); Ikeda, E., et al.,“Hypoxia-induced transcriptional activation and increased mRNA stabilityof vascular endothelial growth factor in C6 glioma cells”, J. Biol.Chem., Vol. 270, pp. 19761-19766 (1995); Levy, A. P., et al.,“Post-transcriptional regulation of vascular endothelial growth factorby hypoxia”, Drug Discov. Today, Vol. 271, pp. 2746-2753 (1996).

Various treatments using VEGF have been suggested (e.g., U.S. Pat. No.5,073,492 issued Dec. 17, 1991; U.S. Pat. No. 5,194,596 issued Mar. 16,1993; and U.S. Pat. No. 5,219,739 issued Jun. 15, 1993) for amelioratingconditions such as myocardial infarction, diabetic ulcers, and surgicalwounds. In particular, several small molecules have been described whichmimic the hypoxic induction of VEGF by activating HIF-1α. However, manyof these molecules, such as cobaltous chloride or deferoxamine cannot beconsidered candidate drug-like molecules because of unfavorablepharmacokinetic characteristics. Still other molecules, such asmersalyl, cannot be considered because of their reactivity. See Agani,F. & Semenza. G. L., “Mersalyl is a novel inducer of vascularendothelial growth factor gene expression and hypoxia-inducible factor 1activity”, Mol. Pharmacol., Vol. 54, pp. 749-754 (1998). Althoughseveral growth factors, such as platelet derived growth factor-BB,transforming growth factor β1, and hepatocyte growth factor, have alsobeen shown to induce VEGF, their effects may be limited to certaintissue types and transformed cell lines and therefore are probably notmediated through HIF-1α. See Brogi, E., et al., “Indirect angiogeniceffect of scatter factor/hepatocyte growth factor via induction ofvascular endothelial growth factor: the case for paracrine amplificationof angiogenesis”, Circulation, Vol. 90, pp. 649-652 (1994); Van, B. E.,et al., “Potentiated angiogenic effect of scatter factor/hepatocytegrowth factor via induction of vascular endothelial growth factor: thecase for paracrine amplification of angiogenesis”, Circulation, Vol. 90,pp. 381-390 (1998). Therefore, there exists a continuing need toidentify classes of compounds that induce VEGF at the transcriptionallevel to increase vascularization of afflicted tissue for the treatmentof the aforementioned disorders.

HIF-1 is a transcription factor that also regulates thehypoxia-inducible EPO gene. HIF-1 binding is required for EPOtranscriptional activation in response to hypoxia. Semenza, G. L.,“Regulation of erythropoietin production: New insights into molecularmechanisms of oxygen homeostasis”, Hematol. Oncol. Clin North Am., Vol.8, pp. 863-884 (1994). In particular, HIF-1α binds to the 3′hypoxia-response element of the EPO gene which results in the markedenhancement of EPO transcription. Semenza, G. L., et al.“Transcriptional regulation of genes encoding glycolytic enzymes byhypoxia-inducible factor 1”, J. Biol. Chem., Vol. 269, pp. 23757-63(1994). EPO, in turn, is essential for maintenance of red blood cells bycontrolling the proliferation and differentiation of erythroidprogenitor cells into red blood cells. Krantz. S. B., “Erythropoietin,”Blood, Vol. 77, pp 419-434 (1991). During fetal development, the liverserves as the primary source of EPO. Shortly before birth, production ofEPO in the liver decreases and the kidney becomes the primary source ofEPO. However, in adults other organs such as the liver and brain producesmall but significant amounts of EPO. A erythropoietin deficiency isassociated with anemia. In humans, the most prevalent form of anemia isassociated with kidney failure.

Compounds have been described that enhance the biosynthesis of EPO suchas those described in U.S. Pat. No. 5,985,913 issued Nov. 16, 1999.Another approach is using injectable recombinant EPO, which is currentlythe therapy of choice for the treatment of anemia due to chronic renalfailure. EPO has been described in the treatment of anemia: associatedwith chemotherapy; that occurs as a consequence of AIDS; and due toprematurity and autologous blood donation. EPO has even been suggestedas a general use agent in pre-operative elective surgery. However, itsextensive use could be limited by high production costs and lack of oralbioavailability. See Qureshi, S. A., et al., “Mimicry of erythropoietinby a nonpeptide molecule”, PNAS, Vol. 96(21) pp. 12156-61 (1999).Therefore, there exists a continuing need for the development of classesof molecules that increase endogenous EPO at the transcriptional levelfor the treatment of the aforementioned disorders.

Thus, it would be advantageous to identify a class of compounds that areeffective in treating hypoxia related disorders.

SUMMARY OF INVENTION

The present invention identifies and provides compounds that areeffective in treating hypoxia related disorders. While not intending tobe limited by theory, it is believed that the compounds herein functionby increasing endogenous EPO and vascularization of tissue in a subjectin need of such treatment. Given the ability of these compounds toinduce EPO, these molecules can be important for the treatment andprophylaxis of anemia associated with kidney disease, as a combinationtherapy with chemotherapy, in preparation for autologous blood donation,and other cases of chronic anemia. Furthermore, given the ability ofthese compounds to increase vascularization in tissue, these moleculescan be important for the treatment of ischemic heart disease, fortreating peripheral vascular disease, and for the enhancement of woundhealing. Other uses of these compounds include: neuroprotection incerebral ischemic conditions; and reducing or preventing hypoxia relateddisorders of cerebral, coronary, or peripheral circulation.

In particular, the present invention relates to compounds having astructure according to Formula (I):

wherein

-   -   (a) R₁ is selected from the group consisting of alkyl, alkene,        alkyne, cycloalkyl. heteroalkyl, aryl, heteroaryl, and        heterocycloalkyl.    -   (b) R₃ is selected from the group consisting of aryl,        heteroaryl, and heterocycloalkyl;    -   (c) R₅ is selected from the group consisting of aryl and        heteroaryl.

This invention also includes optical isomers, diastereomers andenantiomers of the formula above, and pharmaceutically acceptable salts,biohydrolyzable amides, esters, and imides thereof.

The compounds of Formula (I) are useful in the treatment of hypoxiarelated disorders. In particular, the invention provides a method ofincreasing vascularization of tissue in a subject, and a method forincreasing EPO in a subject, by administering the compounds of Formula(I). Accordingly, the invention further provides pharmaceuticalcompositions comprising these compounds.

DETAILED DESCRIPTION OF THE INVENTION I. TERMS AND DEFINITIONS

All percentages, ratios and proportions herein are by weight, unlessotherwise specified.

All documents described herein are hereby incorporated by reference.

When describing the compounds involved in the subject invention, thefollowing terms have the following meanings unless otherwise specified.

“Acyl” or “carbonyl” is a radical formed by removal of the hydroxy froma carboxylic acid (i.e., R—C(═O)—). Preferred acyl groups include (forexample) acetyl, formyl, and propionyl.

“Alkyl” is a saturated hydrocarbon chain having 1 to 15 carbon atoms,preferably 1 to 10. more preferably 1 to 4 carbon atoms. “Alkene” is ahydrocarbon chain having at least one (preferably only one)carbon-carbon double bond and having 2 to 15 carbon atoms, preferably 2to 10, more preferably 2 to 4 carbon atoms. “Alkyne” is a hydrocarbonchain having at least one (preferably only one) carbon-carbon triplebond and having 2 to 15 carbon atoms, preferably 2 to 10, morepreferably 2 to 4 carbon atoms. Alkyl, alkene and alkyne chains(referred to collectively as “hydrocarbon chains”) may be straight orbranched and may be unsubstituted or substituted. Preferred branchedalkyl, alkene and alkyne chains have one or two branches, preferably onebranch. Preferred chains are alkyl. Alkyl, alkene and alkyne hydrocarbonchains each may be unsubstituted or substituted with from 1 to 4substituents; when substituted, preferred chains are mono-, di-, ortri-substituted. Alkyl, alkene and alkyne hydrocarbon chains each may besubstituted with halo, hydroxy, aryloxy (e.g., phenoxy), heteroaryloxy,acyloxy (e.g., acetoxy), carboxy, aryl (e.g., phenyl), heteroaryl,cycloalkyl, heterocycloalkyl, spirocycle, amino, amido, acylamino, keto,thioketo, cyano, or any combination thereof. Preferred hydrocarbongroups include methyl, ethyl, propyl, isopropyl, butyl, vinyl, allyl,butenyl, and exomethylenyl.

Also, as referred to herein, a “lower” alkyl, alkene or alkyne moiety(e.g., “lower alkyl”) is a chain comprised of 1 to 6, preferably from 1to 4, carbon atoms in the case of alkyl and 2 to 6, preferably 2 to 4,carbon atoms in the case of alkene and alkyne.

“Alkoxy” is an oxygen radical having a hydrocarbon chain substituent,where the hydrocarbon chain is an alkyl or alkenyl (i.e., —O-alkyl or—O-alkenyl). Preferred alkoxy groups include (for example) methoxy,ethoxy, propoxy and allyloxy.

“Aryl” is an aromatic hydrocarbon ring. Aryl rings are monocyclic orfused bicyclic ring systems. Monocyclic aryl rings contain from 5 toabout 9 atoms, preferably from 5 to 7 atoms, most preferably from 5 to 6atoms, especially 6 carbon atoms in the ring. Six carbon atom ringmembered monocyclic aryl rings are also referred to as phenyl rings.Bicyclic aryl rings contain from 8 to 17 carbon atoms, preferably 9 to12 carbon atoms, in the ring. Bicyclic aryl rings include ring systemswherein one ring is aryl and the other ring is aryl, cycloalkyl, orheterocycloakyl. Preferred bicyclic aryl rings comprise 5-, 6- or7-membered rings fused to 5-, 6-, or 7-membered rings. Aryl rings may beunsubstituted or substituted with from 1 to 4 substituents on the ring.Aryl may be substituted with halo, cyano, nitro, hydroxy, carboxy,amino, acylamino, alkyl, heteroalkyl, haloalkyl, phenyl, aryloxy,alkoxy, heteroalkyloxy, carbamyl, haloalkyl, methylenedioxy,heteroaryloxy, or any combination thereof. Preferred aryl rings includenaphthyl, tolyl, xylyl, and phenyl. The most preferred aryl ring radicalis phenyl.

“Aryloxy” is an oxygen radical having an aryl substituent (i.e.,—O-aryl). Preferred aryloxy groups include (for example) phenoxy,napthyloxy, methoxyphenoxy, and methylenedioxyphenoxy.

“Cycloalkyl” is a saturated or unsaturated hydrocarbon ring. Cycloalkylrings are not aromatic. Cycloalkyl rings are monocyclic, or are fusedspiro, or bridged bicyclic ring systems. Monocyclic cycloalkyl ringscontain from about 3 to about 9 carbon atoms, preferably from 3 to 7carbon atoms, in the ring. Bicyclic cycloalkyl rings contain from 7 to17 carbon atoms, preferably from 7 to 12 carbon atoms, in the ring.Preferred bicyclic cycloalkyl rings comprise 4-, 5-, 6- or 7-memberedrings fused to 5-, 6-, or 7-membered rings. Cycloalkyl rings may beunsubstituted or substituted with from 1 to 4 substituents on the ring.Cycloalkyl may be substituted with halo, cyano, alkyl, heteroalkyl,haloalkyl, phenyl, keto, hydroxy, carboxy, amino, acylamino, aryloxy,heteroaryloxy, or any combination thereof. Preferred cycloalkyl ringsinclude cyclopropyl, cyclopentyl, and cyclohexyl.

“Halo” or “halogen” is fluoro, chloro, bromo or iodo. Preferred halo arefluoro, chloro and bromo; more preferred typically are chloro andfluoro, especially fluoro.

“Haloalkyl” is a straight, branched, or cyclic hydrocarbon substitutedwith one or more halo substituents. Preferred are C₁-C₁₂ haloalkyls;more preferred are C₁-C₆ haloalkyls; still more preferred still areC₁-C₃ haloalkyls. Preferred halo substituents are fluoro and chloro. Themost preferred haloalkyl is trifluoromethyl.

“Heteroatom” is a nitrogen, sulfur, or oxygen atom. Groups containingmore than one heteroatom may contain different heteroatoms.

“Heteroalkyl” is a saturated or unsaturated chain containing carbon andat least one heteroatom, wherein no two heteroatoms are adjacent.Heteroalkyl chains contain from 2 to 15 member atoms (carbon andheteroatoms) in the chain, preferably 2 to 10, more preferably 2 to 5.For example, alkoxy (i.e., —O-alkyl or —O-heteroalkyl) radicals areincluded in heteroalkyl. Heteroalkyl chains may be straight or branched.Preferred branched heteroalkyl have one or two branches, preferably onebranch. Preferred heteroalkyl are saturated. Unsaturated heteroalkylhave one or more carbon-carbon double bonds and/or one or morecarbon-carbon triple bonds. Preferred unsaturated heteroalkyls have oneor two double bonds or one triple bond, more preferably one double bond.Heteroalkyl chains may be unsubstituted or substituted with from 1 to 4substituents. Preferred substituted heteroalkyl are mono-, di-, ortri-substituted. Heteroalkyl may be substituted with lower alkyl,haloalkyl, halo, hydroxy, aryloxy, heteroaryloxy, acyloxy, carboxy,monocyclic aryl, heteroaryl, cycloalkyl, heterocycloalkyl, spirocycle,amino, acylamino, amido, keto, thioketo, cyano, or any combinationthereof.

“Heteroaryl” is an aromatic ring containing carbon atoms and from 1 toabout 6 heteroatoms in the ring. Heteroaryl rings are monocyclic orfused bicyclic ring systems. Monocyclic heteroaryl rings contain fromabout 5 to about 9 member atoms (carbon and heteroatoms), preferably 5or 6 member atoms, in the ring. Bicyclic heteroaryl rings contain from 8to 17 member atoms, preferably 8 to 12 member atoms, in the ring.Bicyclic heteroaryl rings include ring systems wherein one ring isheteroaryl and the other ring is aryl, heteroaryl, cycloalkyl, orheterocycloalkyl. Preferred bicyclic heteroaryl ring systems comprise5-, 6- or 7-membered rings fused to 5-, 6-, or 7-membered rings.Heteroaryl rings may be unsubstituted or substituted with from 1 to 4substituents on the ring. Heteroaryl may be substituted with halo,cyano, nitro, hydroxy, carboxy, amino, acylamino, alkyl, heteroalkyl,haloalkyl, phenyl, alkoxy, aryloxy, heteroaryloxy, or any combinationthereof. Preferred heteroaryl rings include, but are not limited to, thefollowing:

“Heteroaryloxy” is an oxygen radical having a heteroaryl substituent(i.e., —O-heteroaryl). Preferred heteroaryloxy groups include (forexample) pyridyloxy, furanyloxy, (thiophene)oxy, (oxazole)oxy,(thiazole)oxy, (isoxazole)oxy, pyrmidinyloxy, pyrazinyloxy, andbenzothiazolyloxy.

“Heterocycloalkyl” is a saturated or unsaturated ring containing carbonatoms and from 1 to about 4 (preferably 1 to 3) heteroatoms in the ring.Heterocycloalkyl rings are not aromatic. Heterocycloalkyl rings aremonocyclic, or are fused, bridged, or spiro bicyclic ring systems.Monocyclic heterocycloalkyl rings contain from about 3 to about 9 memberatoms (carbon and heteroatoms), preferably from 5 to 7 member atoms, inthe ring. Bicyclic heterocycloalkyl rings contain from 7 to 17 memberatoms, preferably 7 to 12 member atoms, in the ring. Bicyclicheterocycloalkyl rings contain from about 7 to about 17 ring atoms,preferably from 7 to 12 ring atoms. Bicyclic heterocycloalkyl rings maybe fused, spiro, or bridged ring systems. Preferred bicyclicheterocycloalkyl rings comprise 5-, 6- or 7-membered rings fused to 5-,6-, or 7-membered rings. Heterocycloalkyl rings may be unsubstituted orsubstituted with from 1 to 4 substituents on the ring. Heterocycloalkylmay be substituted with halo, cyano, hydroxy, carboxy, keto, thioketo,amino, acylamino, acyl, amido, alkyl, heteroalkyl, haloalkyl, phenyl,alkoxy, aryloxy or any combination thereof. Preferred substituents onheterocycloalkyl include halo and haloalkyl. Preferred heterocycloalkylrings include, but are not limited to, the following:

“Spirocycle” is an alkyl or heteroalkyl diradical substituent of alkylor heteroalkyl wherein said diradical substituent is attached seminallyand wherein said diradical substituent forms a ring, said ringcontaining 4 to 8 member atoms (carbon or heteroatom), preferably 5 or 6member atoms.

A “pharmaceutically-acceptable salt” is a cationic salt formed at anyacidic (e.g., hydroxamic or carboxylic acid) group, or an anionic saltformed at any basic (e.g., amino) group. Many such salts are known inthe art, as described in World Patent Publication 87/05297, Johnston, etal., published Sep. 11, 1987, incorporated by reference herein.Preferred cationic salts include the alkali metal salts (such as sodiumand potassium), and alkaline earth metal salts (such as magnesium andcalcium) and organic salts. Preferred anionic salts include the halides(such as chloride salts), sulfonates, carboxylates, phosphates, and thelike.

Such salts are well understood by the skilled artisan, and the skilledartisan is able to prepare any number of salts given the knowledge inthe art. Furthermore, it is recognized that the skilled artisan mayprefer one salt over another for reasons of solubility, stability,formulation ease and the like. Determination and optimization of suchsalts is within the purview of the skilled artisan's practice.

A “biohydrolyzable amide” is an amide of a hydroxamic acid-containingcompound of Formula (I) that does not interfere with the vascularizingor EPO increasing activity of these compounds, or that is readilyconverted in vivo by an animal, preferably a mammal, more preferably ahuman subject, to yield an active compound of Formula I. Examples ofsuch amide derivatives are alkoxyamides, where the hydroxyl hydrogen ofthe hydroxamic acid of Formula (I) is replaced by an alkyl moiety, andacyloxyamides, where the hydroxyl hydrogen is replaced by an acyl moiety(i.e., R—C(═O)—).

A “biohydrolyzable hydroxy imide” is an imide of a hydroxamicacid-containing compound of Formula (I) that does not interfere with thevascularizing or EPO increasing activity of these compounds, or that isreadily converted in vivo by an animal, preferably a mammal, morepreferably a human subject to yield an active compound of Formula (I).Examples of such imide derivatives are those where the amino hydrogen ofthe hydroxamic acid of Formula (I) is replaced by an acyl moiety (i.e.,R—C(═O)—).

A “biohydrolyzable ester” is an ester of a hydroxy-containing compoundof Formula (I) that does not interfere with the vascularizing or EPOincreasing activity of these compounds or that is readily converted invivo by an animal to yield an active compound of Formula (I). Suchesters include lower alkyl esters, lower acyloxy-alkyl esters (such asacetoxymethyl, acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyland pivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline estersand alkyl acylamino alkyl esters (such as acetamidomethyl esters).

The terms “optical isomer”, “stereoisomer”, and “diastereomer” have thestandard an recognized meanings (see, e.g., Hawley's Condensed ChemicalDictionary, 11th Ed.). The illustration of specific protected forms andother derivatives of the compounds of the instant invention is notintended to be limiting. The application of other useful protectinggroups, salt forms, etc. is within the ability of the skilled artisan.

II. COMPOUNDS

The invention relates to the method of using the compounds of Formula(I):

where R₁, R₃, and R₅ have the meanings described above. The followingprovides a description of particularly preferred moieties, but is notintended to limit the scope of the claims.

R₁ is selected from the group consisting of: alkyl, alkene, alkyne,cycloalkyl, heteroalkyl, aryl, heteroaryl, and heterocycloalkyl. When R₁is a heteroaryl, R₁ in one mode has a heteroatom in the 2-position. R₁may be 2-pyridine.

R₃ is selected from the group consisting of aryl, heteroaryl, andheterocycloalkyl. In one mode, R₃ is aryl or heteroaryl. In another modeR₃ is aryl substituted in the 2-position with at least hydroxy. In stillanother mode, R₃ is further selected from the group consisting of2-pyridyl, phenyl, 2-hydroxyphenyl, 3-naphthyl.

R₅ is selected from the group consisting of aryl and heteroaryl. In onemode R₅ is further selected from the group consisting of phenyl, andm-chlorophenyl.

III. COMPOUND PREPARATION

The compounds of the invention can be prepared using a variety ofprocedures. The starting materials used in preparing the compounds ofthe invention are known, made by known methods, or are commerciallyavailable. A particularly preferred synthesis is described in thefollowing general reaction scheme. Specific examples for making thecompounds of the present invention are set forth in Section VII, below.

In the general reaction scheme, R₁, R₃, and R₅ are defined above. S1aand S1b starting materials are generally commercially available (such asfrom Aldrich, TCI, or Lancaster). Some di-aromatic enones or chalconesare generally commercially available (such as from Aldrich, TCI, orPfaltz and Bauer). Hydrazine derivatives are generally commerciallyavailable (such as from Aldrich, Pfatz & Bauer, or Lancaster). Compoundsdescribed by S1d, that is Formula (I) can be made by methods known toone of ordinary skill in the art, as described by the following and incited references.

In the above general reaction scheme, the aryl or heteroaryl ketonedepicted as S1a is reacted with an aryl or heteroaryl aldehyde depictedas S1b in a solvent that will allow the formation of the enone to takeplace. Preferred solvents include small (C1-C4) aliphatic alcohols thatmay or may not be wet with water, or a higher boiling ether such astetrahydrofuran. The more preferred solvent is methanol or ethanol. Themost preferred solvent is ethanol. Subsequently, a base is added to thereaction. Preferred bases include but are not limited to methoxides orhydroxides. A preferred base is sodium hydroxide. The reaction isallowed to proceed at a temperature preferably between about −50° C. andabout 75° C., more preferably between about −20° C. and about 45° C.,and most preferred between about 0° C. and about 25° C. The reactiontime is preferably between about 1 and about 24 hours, more preferablybetween about 4 and about 20 hours, and the most preferred between about8 and about 16 hours. Kohler, E. P.& H. M. Chadwell, Org. Synth., Coll.Vol. 1, 2^(nd) ed., p. 78; and Hollinshead, S. P., Tetrahedron Letters,Vol. 37, pp. 9157-9160 (1996).

The resulting enone depicted as S1c is isolated by methods known tothose of ordinary skill in the art. Such methods include, but are notlimited to, extraction, solvent evaporation, precipitation andfiltration, or by flash chromatography on silica gel (Merck, 230-400mesh) using a mixture of solvents, preferably hexanes and ethyl acetate,or dichloromethane and methanol, the most preferred beingdichloromethane and methanol. Most preferable methods of isolation areprecipitation and filtration, or extraction into organic media, followedby flash column chromatography.

The di-aryl/heteroaryl enone S1c is easily reacted with a variety ofhydrazines, either free base, or the mono- or di-hydrochloride salt asdepicted, for example, in Powers, D. G., et al., Tetrahedron, Vol. 54,pp. 4085-4096 (1998). The most preferred hydrazine is free base. Theenone intermediate S1c is placed in a solvent suitable for the reactionin flasks open to atmospheric pressure, or in tubes that are sealedduring the reaction while heating takes place, such as on a Quest™ 210Organic Synthesizer (Argonaut Technologies) as to allow pressure tobuild in the system. Preferred solvents include small aliphatic alcohols(C1-C4) such as methanol, ethanol, or isopropanol, or dimethylsulfoxide, tetrahydrofuran, or dimethylformamide. More preferredsolvents are small aliphatic alcohols or dimethylsulfoxide. The mostpreferred solvent is ethanol. An amount of base is added dependent onthe species of hydrazine, free base, mono or dihydrochloride salt. Apreferred base is sodium hydroxide.

Addition of hydrazine is followed by heating the reaction. Thecyclization is carried out at a temperature between about 0° C. andabout 140° C. The preferred temperature range is between 30° C. and 110°C. The most preferred range is between 55° C. and 85° C. The preferredtime for reaction completion is about 1 to about 15 hours. The morepreferred reaction time is between about 3 and about 12 hours. The mostpreferred reaction time is between about 5 and about 10 hours.

The 1,3,5-substituted dihydropyrazole noted as S1d is isolated bymethods known to one of ordinary skill in the art. Such methods include,but are not limited to, extraction, solvent evaportion, high pressureliquid chromatography (Gilson HPLC, 5%-95% methanol in water, 9 minutegradient, 210 nm), precipitation followed by filtration, orcrystallization followed by filtration.

These steps may be varied to increase yield of desired product. Theskilled artisan will recognize the judicious choice of reactants,solvents, and temperatures is an important considerations in anysuccessful synthesis. Determination of optimal conditions, etc., isroutine. Thus, the skilled artisan can make a variety of compounds usingthe guidance of the scheme above.

It is recognized that the skilled artisan in the art of organicchemistry can readily carry out standard manipulations of organiccompounds without further direction; that is, it is well within thescope and practice of the skilled artisan to carry out suchmanipulations. These include, but are not limited to, reduction ofcarbonyl compounds to their corresponding alcohols, oxidations ofhydroxyls and the like, acylations, aromatic substitutions, bothelectrophilic and nucleophilic, etherifications, esterification andsaponification and the like. Examples of these manipulations arediscussed in standard texts such as March, Advanced Organic Chemistry(Wiley). Carey and Sundberg, Advanced Organic Chemistry (Vol. 2) andother art that the skilled artisan is aware of.

The skilled artisan will also readily appreciate that certain reactionsare best carried out when another potentially reactive functionality onthe molecule is masked or protected, thus avoiding any undesirable sidereactions and/or increasing the yield of the reaction. Often the skilledartisan utilizes protecting groups to accomplish such increased yieldsor to avoid the undesired reactions. These reactions are found in theliterature and are also well within the scope of the skilled artisan.Examples of many of these manipulations can be found for example in T.Greene, Protecting Groups in Organic Synthesis.

The compounds of the invention may have one or more chiral centers. As aresult, one may selectively prepare one optical isomer, includingdiastereomer and enantiomer, over another, for example by chiralstarting materials, catalysts or solvents, or may prepare bothstereoisomers or both optical isomers, including diastereomers andenantiomers, at once (a racemic mixture). Since the compounds of theinvention may exist as racemic mixtures, mixtures of optical isomers,including diastereomers and enantiomers, or stereoisomers may beseparated using known methods, such as chiral salts, chiralchromatography and the like.

In addition, it is recognized that one optical isomer, includingdiastereomer and enantiomer, or stereoisomer may have favorableproperties over the other. Thus when disclosing and claiming theinvention, when one racemic mixture is disclosed, it is clearlycontemplated that both optical isomers, including diastereomers andenantiomers, or stereoisomers substantially free of the other aredisclosed and claimed as well.

IV. METHODS OF USE

The compounds of present invention increase the biological activity ofHIF-1, thereby increasing the transcription of HIF-1 target genes.Without wishing to be bound by theory, the compounds of the presentinvention are believed to increase the biological activity of HIF-1 byone or more of the following mechanisms: i) increasing the mRNAexpression of HIF-1; ii) increasing the expression of the protein HIF-1;iii) enhancing the nuclear localization of HSF-1; and iv) enhancing thetransactivation of HIF-1. Semenza, J. Appl. Physiol., Vol. 88, page 1476(2000). The increased biological activity HIF-1, in turn, leads to theincreased expression of a number of HIF-1 target genes. A non-exhaustivelist of HIF-1 target genes include: adenylate kinase 3,_(α1B)-adrenergic receptor, adrenomedullin, aldolase A, aldolase C,endothelin-1, enolase 1, EPO, glucose transporter 1, glucose transporter3, glyceraldehyde phosphate dehydrogenase, heme oxygenase-1, hexokinase1, hexokinase 2, insulin-like growth factor II, IGF binding protein 1,IGF factor binding protein 3, lactate dehydrogenase A, nitric oxidesynthase 2, p21, p35srj, phosphofructokinase L, phosphoglycerate kinase1, pyruvate kinase M, transferrin, transferrin receptor, VEGF, VEGFreceptor FLT-1. Semenza, J. Appl. Physiol., Vol. 88, pp. 1474-1480(2000).

By increasing the transcription of these HIF-1 target genes, thecompounds of the present invention provide a method of increasing thevascularization of tissue in a subject. As used herein, “vascularizationof tissue” means a pro-angiogenic response whereby blood vessels orother vessels or ducts develop at or around the afflicted tissue. Theafflicted tissue need not be hypoxic or ischemic per se, but rather thecompounds Formula (I) mimic the body's pro-angiogenic response tohypoxia. A non-limiting example of “vascularization” includes capillaryproliferation in a non-healing wound or along the border of ischemictissue. Thus, these compounds enhance the ability of the body torevsacularize damaged tissues or increase vasculature (e.g. to preventhypoxic damage). Non-limiting examples of “tissue” include: cardiactissue, such as myocardium and cardiac ventricles; skeletal muscle;neurological tissue, such as from the cerebellum; internal organs, suchas the stomach, intestine, pancreas, liver, spleen, and lung; and distalappendages such as fingers and toes.

The subject population that would benefit from treatment with thesecompounds is large and includes any subjects requiring pro-angiogenictreatment or recovery from endothelial cell damage or loss. Exampleswould be subjects with hypoxia/ischemia-related tissue damage orcoronary, cerebral, or peripheral arterial disease. Further examplesinclude those subjects with atherosclerosis, those with diabeticpathology including chronic skin lesions, any subject with bonefractures or wounds that do not heal readily, subjects recovering fromsurgeries that require rapid revascularization of affected areas orwhere endothelium is damaged (e.g., vascular graft surgery, balloonangioplasty) or many surgically-related conditions (that is, conditionsthat often lead to surgery and are caused by surgery) such as oralulcers, peptic ulcers, Crohn's disease, skin grafts, and wound healing,or those with conditions such as frostbite, gangrene, erectiledysfunction, hair loss, or poor circulation. Still further examplesinclude those subjects presenting with transient ischemic attacks orangina. The compounds of the present invention may also be involved inextra vascularization, where surrounding tissue needs to be broken downto allow new blood vessels such as in angiofibroma and hemangioma.

Vascularization of tissue can be measured by any person skilled in theart using standard techniques. Non-limiting examples of measuringvascularization in a subject include: SPECT (single photon emissioncomputed tomography); PET (positron emission tomography); MRI (magneticresonance imaging) and combinations thereof, by measuring blood flow tothe tissue before and after treatment. These and other techniques arediscussed in Simons, et al., “Clinical trials in coronary angiogenesis”,Circulation, Vol. 102, pp.73-86 (2000) incorporated herein by reference.

By increasing the transcription of these HIF-1 target genes, thecompounds of the present invention also provide a method of increasingEPO in a subject. EPO transcription is subject to physiologicalregulation at the level of gene transcription in response to hypoxia aprocess that can be mimicked by the compounds of the present inventionby increasing the biological activity of the transcription factor HIF-1.Thus, these compounds enhance the ability of the body to increase EPO.

The subject population that would benefit from treatment with thesecompounds is also large and includes any subjects exhibiting anerythropoietin deficiency. As used herein, “erythropoietin deficiency,”refers to those conditions in which a subject exhibits either a belownormal hematocrit and a below normal level of EPO, or a below normalhematocrit and an average level of EPO, or a normal hematocrit and abelow normal EPO. Any person skilled in the art, using standard methods,can measure the hematocrit and EPO levels in blood. A non-limitingexample of measuring EPO includes an EPO-ELISA kit from R&D Systems(catalogue #DEP00, Minneapolis, Minn.). Another example of measuring EPOincludes a competitive radioimmunoassay as described by Garcia, et al.,“Radioimmunoassay of erythropoietin,” Blood Cells, Vol. 5, pp. 405-419(1979) incorporated herein by reference.

Examples of a subject population exhibiting an erythropoietin deficiencyinclude those subjects exhibiting an erythropoietin deficiencyassociated with anemia. In another example, the erythropoietindeficiency is associated with anemia due to chronic renal failure. Othernon-limiting examples of other deficiencies associated with anemiainclude, but are not limited to, anemia due to: prematurity; autologousblood donation; chronic infection; rheumatoid arthritis; AIDS;AZT-treated HIV-infection; malignancies; stem cell therapy; and anemiaassociated with: irritable-bowel disease; hypothyroidism; malnutrition;chemotherapy; and bone marrow transplantation.

Though not essential for activity or efficacy, certain diseases,disorders, and unwanted conditions preferably are treated with compoundsthat act on the afflicted tissues or regions of the body. For example, acompound that displays a higher degree of affinity for the heart wouldbe preferred for treatment of ischemic heart disease by increasingvascularization to the cardiac tissue than other compounds that are lessspecific.

In addition, certain compounds are more bioavailable to certain tissuesthat others. Choosing a compound which is more bioavailable to a certaintissue and which acts on the hypoxia-related disorder found in thattissue provides for the specific treatment of the disease, disorder, orunwanted condition. For example, compounds of this invention may vary intheir ability to penetrate in neurological tissue. Thus, compounds maybe selected for neurological protection from hypoxia (e.g., stroke) byincreasing vascularization to the neurological tissue. See, e.g.,Bergeron, S., et al., “Role of hypoxia-inducible factor-1 inhypoxia-induced ischemic tolerance in neonatal rat brain,” Ann. Neuro.,Vol. 48(3), pp. 285-96, (2000); Marti, H. J., et al., “Hypoxia-inducedvascular endothelial growth factor expression precedesneovascularization after cerebral ischemia,” Am. J. Pathol., Vol.156(3), pp. 965-76 (2000).

Determination of the specificity of a compound to a particular type oftissue is within the skill of the artisan in that field. For example, ifincreasing EPO in an adult subject is the therapeutic goal, the abilityof compounds of the present invention to increase EPO production inkidney cells can be screened by their ability to increase blood plasmalevel of EPO via a radioimmunoassay (e.g., DiaSorin).

The compounds of this invention are also useful for prophylactic oracute treatment. They are administered in any way the skilled artisan inthe fields of medicine or pharmacology would desire. It is immediatelyapparent to the skilled artisan that preferred routes of administrationwould depend upon the disease state being treated and the dosage formchosen. Preferred routes for systemic administration includeadministration perorally or parenterally.

However, the skilled artisan will readily appreciate the advantage ofadministering the compounds of the present invention directly to theaffected area for many diseases, disorders, or unwanted conditions. Forexample, given the compounds of the present invention increasevascularization of tissue, it may be advantageous to administer thecompounds directly to the area of the tissue in need of vascularizationsuch as in the area affected by surgical trauma (e. g., angioplasty),non-healing wound (e.g., topical to the skin), or for opthalmic andperiodontal indications.

V. COMPOSITIONS

The compositions of the invention comprise:

-   -   (a) a safe and effective amount of a compound of the invention;        and    -   (b) a pharmaceutically-acceptable carrier.

As discussed above, hypoxia plays an important role in the pathogenesisof many diseases and disorders. The compounds of the present inventionare useful in therapy with regard to these hypoxia related conditions aswell a treating tissue in need of pro-angiogenic therapy and increasingEPO.

The invention compounds can therefore be formulated into pharmaceuticalcompositions for use in increasing vascularization of tissue andincreasing EPO. Standard pharmaceutical formulation techniques are used,such as those disclosed in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., latest edition.

A “safe and effective amount” of a Formula (I) compound is an amountthat is effective, to increase vascularization and/or increase EPO atthe site(s) of activity, in an animal, preferably a mammal, morepreferably a human subject, without undue adverse side effects (such astoxicity, irritation, or allergic response), commensurate with areasonable benefit/risk ratio when used in the manner or this invention.The specific “safe and effective amount” will, obviously, vary with suchfactors as the particular condition being treated, the physicalcondition of the patient, the duration of treatment, the nature ofconcurrent therapy (if any), the specific dosage form to be used, thecarrier employed, the solubility of the Formula (I) compound therein,and the dosage regimen desired for the composition.

In addition to the subject compound, the compositions of the subjectinvention contain a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier”, as used herein, means one or morecompatible solid or liquid filler diluents or encapsulating substanceswhich are suitable for administration to an animal, preferably a mammal,more preferably a human. The term “compatible”, as used herein, meansthat the components of the composition are capable of being commingledwith the subject compound, and with each other, in a manner such thatthere is no interaction that would substantially reduce thepharmaceutical efficacy of the composition under ordinary usesituations. Pharmaceutically-acceptable carriers must, of course, be ofsufficiently high purity and sufficiently low toxicity to render themsuitable for administration to the animal, preferably a mammal, morepreferably a human being treated.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tweens®; wetting agents, such sodium buryl sulfate; coloring agents;flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the subject compound is basically determined by the waythe compound is to be administered.

If the subject compound is to be injected, the preferredpharmaceutically-acceptable carrier is sterile, physiological saline,with a blood-compatible colloidal suspending agent, the pH of which hasbeen adjusted to about 7.4.

In particular, pharmaceutically-acceptable carriers for systemicadministration include sugars, starches, cellulose and its derivatives,malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils,polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonicsaline, and pyrogen-free water. Preferred carriers for parenteraladministration include propylene glycol, ethyl oleate, pyrrolidone,ethanol, and sesame oil. Preferably, the pharmaceutically-acceptablecarrier, in compositions for parenteral administration, comprises atleast about 90% by weight of the total composition.

The compositions of this invention are preferably provided in unitdosage form. As used herein, a “unit dosage form” is a composition ofthis invention containing an amount of a Formula (I) compound that issuitable for administration to an animal, preferably a mammal, morepreferably a human subject, in a single dose, according to good medicalpractice. These compositions preferably contain from about 5 mg(milligrams) to about 1000 mg, more preferably from about 10 mg to about500 mg, more preferably from about 10 mg to about 300 mg, of a Formula(I) compound.

The compositions of this invention may be in any of a variety of forms,suitable, for example, for oral, rectal, topical, nasal, ocular orparenteral administration. Depending upon the particular route ofadministration desired, a variety of pharmaceutically-acceptablecarriers well-known in the art may be used. These include solid orliquid fillers, diluents, hydrotropes, surface-active agents, andencapsulating substances. Optional pharmaceutically-active materials maybe included, which do not substantially interfere with the inhibitoryactivity of the Formula (I) compound. The amount of carrier employed inconjunction with the Formula (I) compound is sufficient to provide apractical quantity of material for administration per unit dose of theFormula (I) compound. Techniques and compositions for making dosageforms useful in the methods of this invention are described in thefollowing references, all incorporated by reference herein: ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, editors, 1979);Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); andAnsel, Introduction to Pharmaceutical Dosage Forms 2d Edition (1976).

Various oral dosage forms can be used including such solid forms astablets. capsules, granules and bulk powders. These oral forms comprisea safe and effective amount, usually at least about 5%, and preferablyfrom about 25% to about 50%, of the Formula (I) compound. Tablets can becompressed, tablet triturates, enteric-coated, sugar-coated,film-coated, or multiple-compressed, containing suitable binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, flow-inducing agents, and melting agents. Liquid oral dosageforms include aqueous solutions, emulsions, suspensions, solutionsand/or suspensions reconstituted from non-effervescent granules, andeffervescent preparations reconstituted from effervescent granules, andcontaining suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, melting agents, coloring agentsand flavoring agents.

The pharmaceutically-acceptable carrier suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical for the purposes of the subjectinvention, and can be readily made by a person skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, Avicel® RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject compound isreleased in the gastrointestinal tract in the vicinity of the desiredtopical application, or at various times to extend he desired action.Such dosage forms typically include, but are not limited to, one or moreof cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragit®coatings, waxes and shellac.

Compositions of the subject invention may optionally include other drugactives.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal, suppository, and nasal dosageforms. Such compositions typically comprise one or more of solublefiller substances such as sucrose, sorbitol and mannitol; and binderssuch as acacia, microcrystalline cellulose, carboxymethyl cellulose andhydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners,colorants, antioxidants and flavoring agents disclosed above may also beincluded.

The compositions of this invention can also be administered topically toa subject, e.g., by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch”. Such compositions include, for example, lotions, creams,solutions, gels and solids. These topical compositions preferablycomprise a safe and effective amount, usually at least about 0.1%, andpreferably from about 1% to about 5%, of the Formula (I) compound.Suitable carriers for topical administration preferably remain in placeon the skin as a continuous film, and resist being removed byperspiration or immersion in water. Generally, the carrier is organic innature and capable of having dispersed or dissolved therein the Formula(I) compound. The carrier may include pharmaceutically-acceptableemollients, emulsifiers, thickening agents, solvents and the like.

VI. METHODS OF ADMINISTRATION

This invention also provides methods of increasing vascularization oftissue and/or increasing EPO in a human or other animal subject, byadministering a safe and effective amount of a Formula (I) compound tosaid subject. The methods of the invention are useful in treating orpreventing disorders described above.

Compositions of this invention can be administered topically orsystemically. Systemic application includes any method of introducingFormula (I) compound into the tissues of the body, e.g.,intra-articular, intrathecal, epidural, intramuscular, transdermal,intravenous, intraperitoneal, subcutaneous, sublingual, rectal, ocular,and oral administration. The Formula (I) compounds of the presentinvention are preferably administered orally.

The specific dosage of inhibitor to be administered, as well as theduration of treatment, and whether the treatment is topical or systemic,are interdependent. The dosage and treatment regimen will also dependupon such factors as the specific Formula (I) compound used, thetreatment indication, the ability of the Formula (I) compound to reachminimum inhibitory concentrations to vascularize affected tissue or toincrease EPO to desired level, and the personal attributes of thesubject (such as weight), compliance with the treatment regimen, and thepresence and severity of any side effects of the treatment.

When determining systemic dosage of a compound of Formula (I) whereinthe therapeutic goal is to increase vascularization in tissue, anysynergistic interactions of the compound with endogenous eventsoccurring in the injured tissue will be taken into account to avoidundesired effects in non-injured tissues. To the extent (if any) thereis synergy between endogenous responses to moderate degrees of hypoxiaand the compounds of the present invention, systemic administration canbe used to generate a tissue specific response. In this manner,angiogenesis would be stimulated in tissues where it is required andpotentially harmful neovascularization (e.g., proliferative retinopathy)in already well-vascularized tissues can be controlled or avoided.

Typically, for a human adult (weighing approximately 70 kilograms), fromabout 5 mg to about 3000 mg, more preferably from about 5 mg to about1000 mg, more preferably from about 10 ma to about 100 mg, of Formula(I) compound are administered per day for systemic administration. It isunderstood that these dosage ranges are by way of example only, and thatdaily administration can be adjusted depending on the factors listedabove.

A preferred method of systemic administration is oral. Individual dosesof from bout 10 mg to about 1000 mg, preferably from about 10 mg toabout 300 mg, are preferred.

Topical administration can be used to deliver the Formula (I) compoundsystemically, or to treat a subject locally. The amounts of Formula (I)compound to be topically administered depends upon such factors as skinsensitivity, type and location of the tissue to be treated thecomposition and carrier (if any) to be administered, the particularFormula (I) compound to be administered, as well as the particulardisorder to be treated and the extent to which systemic (asdistinguished from local) effects are desired.

The compounds of the present invention can be targeted to specificlocations within the body by using targeting ligands well known in theart. For example, to focus a Formula (I) compound to ischemic cardiactissue, the compound is conjugated to an antibody or fragment thereofwhich is immunoreactive with a cardiac cell marker as is broadlyunderstood in the preparation of immunopharmaceuticals in general. Thetargeting ligand can also be a ligand suitable for a receptor that ispresent on the cardiac tissue. Any targeting ligand that specificallyreacts with a marker for the intended tweet tissue can be used. Methodsfor coupling the invention compound to the targeting ligand are wellknown and are similar to those described below for coupling to acarrier. The conjugates are formulated and administered as describedabove.

For localized conditions, topical administration is preferred. Forexample, to treat a non-healing skin lesion, the compound is appliedlocally and topically, in a gel, paste, salve or ointment. For treatmentof oral diseases, such as gingivitis, the compound may be appliedlocally in a gel, paste, mouth wash, or implant. The mode of treatmentthus reflects the nature of the condition and suitable formulations forany selected route are available in the art.

In all of the foregoing, of course, the compounds of the invention canbe administered alone or as mixtures, and the compositions may furtherinclude additional drugs or excipients as appropriate for theindication.

VII. EXAMPLES Compound Preparation Examples 1-20

The following chart shows the structure of compounds made according tothe procedures described in Examples 1-20.

TABLE I Example R1 R3 R5 EC₅₀ ¹ 1. phenyl 2-pyridyl phenyl 23 2.2-pyridyl phenyl phenyl 6 3. 2-pyridyl 2-hydroxyphenyl phenyl 1.7 4.1-benzothiazole 2-hydroxyphenyl phenyl 27 5. 2-pyridyl 2-hydroxyphenylp-toluyl 5.7 6. 2-pyridyl 2-hydroxyphenyl p-methoxyphenyl 5.68 7.2-pyridyl 2-hydroxyphenyl o-methoxyphenyl 5.5 8. 2-pyridyl2-hydroxyphenyl 2-pyridyl 5.4 9. 2-pyridyl 2-hydroxyphenylo-hydroxyphenyl 5.4 10. methyl 2-hydroxyphenyl 2-pyridyl 92 11. methyl2-hydroxyphenyl 1-thiazole 12 12. 2-hydroxyethyl 2-hydroxyphenyl2-pyridyl 65 13. 2-cyanoethyl 2-hydroxyphenyl 2-pyridyl 18 14. p-toluyl2-pyridyl phenyl 2 15. p-bromophenyl 2-pyridyl phenyl 6.3 16. phenyl2-pyridyl m-chlorophenyl 1.7 17. phenyl phenyl p-dimethylaminophenyl 618. methyl 3-naphthyl p-methoxyphenyl 11 19. methyl 3-naphthylp-fluorophenyl 3.6 20. methyl 3-naphthyl p-trifluoromethylphenyl 13¹EC₅₀ is the concentration of compound that induces the production of anamount of VEGF equal to half the maximum amount of VEGF induced by thecompound.

Compounds are analyzed using ¹H and ¹³C NMR obtained on a Varian Unityplus 300 MHz spectrometer, chemical shifts are reported in δ ppmdownfield from TMS as an internal standard. The compounds are alsoanalyzed using elemental analysis, mass spectra using a FisonsPlatform-II quadrupole mass spectrometer, high resolution mass spectraand/or IR spectra as appropriate. Thin layer chromatography (hereinafter“TLC”) analysis is performed on glass mounted silica gel plates (200-300mesh; Baker or Analtech) with fluorescent indicator and visualized usingUV detection.

Example 1 2-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-pyridine

3-Phenyl-1-pyridin-2-yl-propenone (1c)

Method A: To solution of 2-acetylpyridine 1a (10 mmol, 1 equiv.) andtetrahydrofuran (25 mL) is added benzaldehyde 1b (10 mmol, 1 equiv.).Sodium methoxide (12 mmol, 1.2 equiv.) in methanol (24 mL, 0.5M) isintroduced into the reaction and is agitated for 15 hours at roomtemperature. Hydrochloric acid (1N) is added until a neutral pH isobtained. The solution is extracted with dichloromethane twice, andwashed once with water. The combined organic layers are dried overMgSO₄, and concentrated under reduced pressure. The residue is purifiedby flash column chromatography on SiO₂ (33% ethyl acetate/hexanes) togive the 3-phenyl-1-pyridin-2-yl-propenone 1c as a pale yellow solid.See Hollinshead, S. P., Tetrahedron Letters, Vol 37, pp. 9157-9160(1996).

Method B: A solution of NaOH (12.8 mmol, 1.28 equiv.) in water (4.55 g)and ethanol (2.32 g) are introduced into a round bottom flask with amagnetic stirrer. Into the alkaline solution is placed 2-acetyl pyridine1a (10 mmol, 1 equiv.) and the flask is rapidly surrounded by ice andthe stirrer is started. The temperature reaches 10° C.-15° C. andbenzaldehyde 1b (10 mmol, 1 equiv.) is added at once. The ice bath isremoved and the reaction is stirred for 15 hours. The reaction isfiltered. The filtrate is concentrated under reduced pressure and theproduct is purified by flash column chromatography on SiO₂ (33% ethylacetate/hexanes) to give the 3-phenyl-1-pyridin-2-yl-propenone 1c as apale yellow solid. See Kohler. E. P. & H. M. Chadwell, H. M., Org.Synth. Coll. Vol. 1, 2^(nd) ed., p. 78.

2-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-pyridine (1d)

To a solution of a 3-phenyl-1-pyridin-2-yl-propenone 1c (0.10 mmol, 1equiv.) in ethanol (4 mL). is added phenyl hydrazine free base (0.10mmol, 1 equiv.) and sodium hydroxide (0.10 mmol. 1 equiv.). The mixtureis stirred at 60° C. for 8 hours (temperature and reaction time canvary, higher temperature if in a sealed system). Water and ethyl acetateis added to the reaction and the phases are separated. The organic layeris washed until the water runs clear, and the aqueous layer is extractedwith ethyl acetate two times. The combined organic layer is dried overNa₂SO₄, decanted and concentrated under reduced pressure. The residue ispurified by high pressure liquid chromatography (Gilson HPLC. 5%-95%methanol in water, 9 minute gradient. 210 nm), and concentrated underreduced pressure to afford2-(1,5-diphenyl4,5-dihydro-1H-pyrazol-3-yl)-pyridine 1d as a yellowpowder. Powers, D. G., et al,. Tetrahedron, Vol. 54, pp. 4085-4096(1998).

Example 2 2-(3,5-Diphenyl-4,5-dihydro-pyrazol-1-yl)-pyridine

Example 2 is prepared using 1-acetylbenzene and benzaldehyde (method A)to afford 1,3-diphenyl-propenone. The propenone is reacted with2-hydrazinopyridine following the procedure described for Example 1.

Example 3 2-(5-Phenyl-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol

Example 3 is prepared using 2-hydroxychalcone and 2-hydrazinopyridinefollowing the procedure described for Example 1.

Example 42-(1-Benzothiazol-2-yl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol

Example 4 is prepared using 2-hydroxychalcone andbenzothiazol-2-yl-hydrazine following the procedure described forExample 1.

Example 52-(1-Pyridin-2-yl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol

Example 5 is prepared using 1-(2-hydroxy-phenyl)-3-p-tolyl-propenone and2-hydrazinopyridine following the procedure described for Example 2.

Example 62-[5-(4-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol

Example 6 is prepared using 1-(2-hydroxy-phenyl)-3-p-methoxy-propenoneand 2-hydrazinopyridine following the procedure described for Example 2.Example 72-[5-(2-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol

Example 7 is prepared using 1-(2-hydroxy-phenyl)-3-o-methoxy-propenoneand 2-hydrazinopyridine following the procedure described for Example 2.

Example 8 2-(1,5-Di-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol

Example 8 is prepared using 2-acetylbenzene and2-pyridinecarboxaladehyde (method A) to afford1-(2-hydroxy-phenyl)-3-pyridin-2-yl-propenone. The propenone is reactedwith 2-hydrazinopyridine following the procedure described for Example1.

Example 92-[5(2-hydroxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol

Example 9 is prepared using 2-acetylbenzene and 2-salicylaldehyde(method A) to afford 1,3-bis-(2-hydroxy-phenyl)-propenone. The propenoneis reacted with and 2-hydrazinopyridine following the proceduredescribed for Example 1.

Example 102-(1-Methyl-5-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol

Example 10 is prepared using1-(2-hydroxy-phenyl)-3-pyridin-2-yl-propenone and methylhydrazinefollowing the procedure described for Example 1.

Example 112-(1-Methyl-5-thiazol-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol

Example 11 is prepared using 2-acetylbenzene and thiazole-2-carbaldehyde(method A) to afford 1-(2-hydroxy-phenyl)-3-thiazol-2-yl-propenone. Thepropenone is reacted with methylhydrazine following the proceduredescribed for Example 1.

Example 122-[1-(2-Hydroxy-ethyl)-5-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol

Example 12 is prepared using1-(2-hydroxy-phenyl)-3-pyridin-2-yl-propenone and 2-hydrazino-ethanolfollowing the procedure described for Example 1.

Example 133-[3-(2-Hydroxy-phenyl)-5-pyridin-2-yl-4,5-dihydro-pyrazol-1-yl]-propionitrile

Example 13 is prepared using1-(2-hydroxy-phenyl)-3-pyridin-2-yl-propenone and3-hydrazino-propionitrile following the procedure described for Example1.

Example 14 2-(5-Phenyl-1-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-pyridine

Purchased from Sigma.

Example 152-[1-(4-Bromo-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-pyridine

Purchased from Sigma.

Example 162-[5-(3-Chloro-phenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-pyridine

Purchased from Sigma.

Example 17[4-(2,5-Diphenyl-3,4-dihydro-2H-pyrazol-3-yl)-phenyl]-dimethyl-amine

Purchased from Sigma.

Example 185-(4-Methoxy-phenyl)-1-methyl-3-naphthalen-2-yl-4,5-dihydro-1H-pyrazole

Purchased from Maybridge.

Example 195-(4-Fluoro-phenyl)-1-methyl-3-naphthalen-2-yl-4,5-dihydro-1H-pyrazole

Purchased from Maybridge.

Example 201-Methyl-3-naphthalen-2-yl-5-(4-trifluoromethyl-phenyl)-4,5-dihydro-1H-pyrazole

Purchased from Maybridge.

VIII. EXAMPLES Methods of Screening Compounds

A compound of Formula (I) can be screened for the appropriatetherapeutic use in a number of different ways. The following assays alsomay be used to optimize therapeutic doses of the compounds. In turn,toxicity would be established according to standard tests well known inthe art for determination of compound toxicity.

Examples I-VI teach methods of screening compounds appropriate for thetherapeutic goal of increasing vascularization of tissue. Although thefollowing examples may focus on the HIF-1 target gene VEGF as a wellknown mediator of angiogenesis, one skilled in the art will appreciatethat these, and other methods, can be readily adapted to screen thecompounds of Formula (I) to other HIF-1 target genes as well as othertherapeutic goals. Indeed, given that compounds increase the biologicalactivity of a transcription factor, HIF-1 controls the expression ofmultiple genes involved in angiogenesis and thus will presumably give asuperior clinical outcome compared to treatment with a single angiogenicfactor such as VEGF. On the other hand, if the therapeutic goal is toincrease EPO, these methods can be adapted to screen for compounds ofFormula (I) that increase the transcription of EPO.

Example I details a VEGF-luciferase reporter gene assay that measures acompound's ability to stimulate transcription of a luciferase genelinked to an upstream VEGF promoter-enhancer sequence. Example IIdetails an enzyme-linked immunosorbancy assay (ELISA) for VEGF thatmeasures the ability of a compound of the present invention to stimulatesecretion of VEGF from a cell. Example III details a gene panel assay.Messenger RNA for eight different genes is measured from a cell linethat is treated with a compound of the present invention. Example IVdetails an immunoblot assay for the induction of HIF-1. Example Vdetails the effect the compounds of the present invention have uponincreased amount of VEGF in rat cardiac tissue. Example VI details thelevel of EPO in blood after rats are treated with the compounds of thepresent invention.

Example I

Any compound of the present invention can be screened by aVEGF-luciferase assay. The assay evaluates the transcriptional activityof the compounds. A VEGF-luciferase cell line is prepared in a ELEMparental line as described in Engelmann, G. L., et al., “Formation offetal rat cardiac cell clones by retroviral transformation: retention ofselect myocyte characteristics”, J. Mol. Cell. Cardio., Vol. 25, pp.2979-84 (1998). ELEM cells are v-H-ras transformed neonatal ratventricular cardiomyocytes. A luciferase reporter plasmid is constructedusing the pGL2-basic (Promega) plasmid containing an insert consistingof 2.65 kb of the vegf promoter (bp −2274 to +379 relative to thetranscription initiation site) fused to the firefly luciferase codingsequences as well as SV40 intron and polyadenylation signals. Thisreporter plasmid, along with a control plasmid containing anSV40-neomyocin and Cytomeagalovirus (CMV)-βgal construct, aretransfected into ELEM cells and stable transfectants are selected inG418. Responses of the VEGF-luciferase reporter to hypoxia and todesferoximine, a mimetic of hypoxic responses, are tested, and a clonewith strong and reproducible responses are selected. ELEM cells andtheir transfectants are grown in Dulbecco's Modified Eagle's Medium/F12(1:1) (Life Technologies) that include 10% heat-inactivated fetal bovineserum, unless otherwise indicated (Life Technologies), a 1×penicillin/streptomysin mixture (Life Technologies) and 500 μg/ml G418(Life Technologies). Tissue culture plates are precoated withfibronectin. The standard condition for assaying is established asfollows: cells are plated in 96-well plates at a density of 6×10³ cellsper well in 100 μl of media per well. 42 hrs later, compounds are addedto the wells in 10 μl aliquots. Six hours after addition of compounds,cells are lysed and assayed for luciferase and β-galactosidase activity.Compounds for pharmaceutical use are selected based upon cellsexhibiting the greatest luciferase and β-galactosidase activity.

Example II

Any compound of the present invention can be screened by a VEGF-ELISAassay. VEGF secretion is assayed in the HL-1 cardiomyocyte cell line asdescribed in Claycomb. W. C., “HL-1 cells: a cardiac muscle cell linethat contracts and retains phenotypic characteristics of the adultcardiomyocyte”, Proc. Natl. Acad. Sci U.S.A., Vol. 95, pp. 2979-84(1998). For experiments, HL-1 cells are plated in 96-well plates at adensity of 2×10⁴ cells per well in growth medium: EXCEL 320 media (JRHBiosciences, Lenexia, Kans.) plus 10% fetal bovine serum (BioWhitaker),100 nM retinoic acid (Sigma, St. Louis, Mo.), 10 μM norepinephrine(Sigma), 10 μg/ml insulin (Life Technologies, Grand Island, N.Y.), 1×non-essential amino acid supplement (Life Technologies), 50 μg/mlendothelial cell growth supplement (Upstate Biotechnology, Lake Placid,N.Y.), 100 units/ml penicillin, 100 units/ml streptomycin (LifeTechnologies). After 24 hrs, the media is replaced with a low-growthmedia (HL-1 media with 2% fetal bovine serum and without endothelialcell growth supplement) containing test compounds with 0.5% dimethylsulfoxide (DMSO). After 18 hrs, conditioned media from the treated cellswas assayed for VEGF content with a Murine VEGF-ELISA kit (R&D Systems,Minneapolis, Minn.). The amount of VEGF secreted into the media over an18 hr period is normalized to positive control wells which are treatedwith a maximally effective concentration (1 mM) of deferoxamine mesylate(Sigma, St. Louis) and are reported as “percent activity,” where theVEGF in unstimulated wells is defined as 0% and the VEGF in wellstreated with 1 mM deferoxamine is defined as 100%. Potency is determinedby stimulating cells with 10 concentrations of test compounds (200 nM to100 μM) and fitting the data to a variable slope sigmoidal dose-responsecurve using GraphPad Prism version 3.00 for Windows 95 (GraphPadSoftware, San Diego, Calif.). Determinations for each test compound areperformed in three separate experiments and the means ± standarddeviation of the Log(EC5) and maximum response are evaluated. Compoundsfor pharmaceutical use are selected based upon the greatest percentactivity.

Example III

Any compound of the present invention can be screened by a geneselectivity panel assay to determine the specificity of induction ofVEGF. Messenger RNA for eight genes expressed in HL-1 cells isquantified using real-time reverse transcriptase-polymerase chainreaction (TaqMan) in duplex reactions using β-actin as a controlreference gene as described in Heid, C. A., et al., “Real timequantitative PCR”, Genome Res., Vol. 6, pp.986-994 (1996). HL-1 cellsare treated for 18 hr with lest compounds at concentrations equal to 10times the EC₅₀, and total RNA is prepared using TriReagent (MolecularResearch Center, Cincinnati, Ohio). Polymerase Chain Reaction (PCR)primers specific to each gene of the panel are designed to span intronswhere possible and are generated using the Primer Express™ Oligo DesignSoftware System (Applied Biosystems) and compared with sequences in theGenBank database to eliminate cross-hybridization with other genes. AllTaqMan probes designed against target genes are 5′-labeled with 6-FAM(6-carboxy-fluorescein) reporter dye and 3′-labeled with TAMRA(6-carboxy-tetramethyl-rhodamine) quencher dye (see Table II). TheTaqMan® probe specific for the β-actin control gene are 5′-labeled withVIC (Applied Biosystems) reporter dye (see Table II) to enable dualamplification and resolution of target and control products in the samereaction for normalization. Primers and probe monocyte chemoattractantprotein-1 (MCP-1) are from Applied Biosystems sold as TaqMan® PDARTarget Reagents.

Primer concentrations are optimized to ensure that both genes in eachreaction are amplified with equal efficiencies and that primers for onegene do not affect the efficiency of amplification of the other. Allgenes are amplified in a reaction mixture containing 15 ng RNA, 3.12 mMmanganese acetate, 1.25 mM deoxyadenine triphosphate (dATP),deoxycytidine triphosphate (dCTP), deoxyguanidine triphosphate (dGTP),2.5 mM deoxyuridine triphosphate (dUTP), 2.5 units rTth DNA Polymerase,1 unit AmpErase UNG, 150 nM target probe and 150 nM β-actin probe. VEGF,transforming growth Factor-β 1 (TGF-β1), sarcolemal endoplasmicreticulum calcium ATPase (SERCA), angiopoietin-1 (Ang-1), and β-myosinheavy chain (β-MHC) RNA are each amplified in separate reactionscontaining the reaction mixture as previously described as well as 300nM forward and reverse target primers and 60 nM β-actin forward andreverse primers. Glyceraldehyde phosphate dehydrogenase (GAPDH),α-myosin heavy chain (α-MHC), and atrial natriuretic peptide (ANP) areeach amplified in separate reactions containing the reaction mixture aspreviously described as well as 80 nM forward and reverse target primersand 60 nM β-actin forward and reverse primers. MCP-1 is amplified usinga 20× primer and probe stock solution prepared and supplied by AppliedBiosystems and is used as 1× in the PCR reaction. PCR Thermal cyclingfor all reactions is 50° C.×2 min. 60° C.×30 min, 95° C.×5 min, followedby 40 cycles of 94° C.×20 seconds and 62° C.×1 min.). Raw CycleThreshold (CT) values for each target gene are calculated by theSequence Detection Software (Applied Biosystems). Each target gene levelis then compared relative to the β-actin level in that same ample bysubtracting the CT for β-actin from the CT for the target gene to arriveat a Δ-CT level. In turn, Δ-CT levels for treated samples are thensubtracted from Δ-CT level of vehicle treated samples to arrive at aΔΔ-CT level for that gene. ΔΔ-CT levels can be converted to a percentchange according to the relationship 100×2^(−(ΔΔCt))=% change. Compoundsfor pharmaceutical use are selected based on having a high % change inVEGF or GAPDH expression and low % change for the non-hypoxia regulatedgenes. TABLE II Primer Sequences Probe Sequence Probe Gene (5′ to 3′)(5′ to 3′) Dye VEGF Forward: ACCATGCCAAGTGGTCCCAGGC FAMACCCTGGCTTTACTGCTGTACCT (SEQ ID NO:3) (SEQ ID NO:1) Reverse:TGGGACTTCTGCTCTCCTTCTG (SEQ ID NO:2) SERC Forward:ACTACAGTCAAACATGCGCTGTGAGAAGCTG FAM A GTAGACAGATGTTGGTGCAATACAAGTA (SEQID NO:6) (SEQ ID NO:4) Reverse: CAATACCTGTTACCAGCACAGAAACT (SEQ ID NO:5)TGF- Forward: CCACGTGGAAATCAACGGGATCAGC FAM β1 GCTCTTGTGACAGCAAAGATAACAA(SEQ ID NO:9) (SEQ ID NO:7) Reverse: GGTCGCCCCGACGTTT (SEQ ID NO:8) ANPForward: ATGGATTTCAAGAACCTGCTAGACCACCTGG FAM TGCGGTGTCCAACACAGATC (SEQID NO: 12) (SEQ ID NO:10) Reverse: GCTTCCTCAGTCTGCTCACTCA (SEQ ID NO:11)β- Forward: CCCAGCTCTAAGGGTGCCCGTGAA FAM MHC GTGCCAAGGGCCTGAATG (SEQ IDNO:15) (SEQ ID NO:13) Reverse: CACCTAAAGGGCTGTTGCAAA (SEQ ID NO:14)α-MHC Forward: ATGTCCCGGCTCTTGGCCCG FAM GGAGGAGAGGGCGGACAT (SEQ IDNO:18) (SEQ ID NO:16) Reverse: AGAGGTTATTCCTCGTCGTGCAT (SEQ ID NO:17)GAPD Forward: CAGAAGACTGTGGATGGCCCCTC FAM H TGCACCACCAACTGCTTAG (SEQ IDNO:21) (SEQ ID NO:19) Reverse: GGATGCAGGGATGATGTFC (SEQ ID NO:20) MCP-1proprietary proprietary FAM (Applied Biosystems) (Applied Biosystems)β-actin Forward: CAGGAGTACGATGAGTCCGGCCCC VIC GTCCACCTTCCAGCAGATGTG (SEQID NO:24) (SEQ ID NO:22) Reverse: CAGTCCGCCTAGAAGCACTTG (SEQ ID NO:23)

Example IV

Any compound of the present invention can be screened by an imnnunoplotassay for the HIF-1α protein. HEK-293 cells are treated with testcompounds for 2 to 18 hrs and nuclear and cytoplasmic extracts are made.Cells are lysed at 4 C in a lysis buffer consisting of: 10 MM Tris HCl,pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5% Np-40, and containing proteaseinhibitors: 10 mM NaF, 1 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml pepstatin,2 mM sodium orthovanadate and crude cytoplasmic fractions were removed.Nuclear pellets were extracted for 20 min at 4 C in 20 mM HEPES, pH 7.9,1 mM EDTA 420 mM NaCl, and 20% glycerol with protease inhibitors and thesupernatants are collected following centrifugation for 15 min at10,000×g. Protein concentration of extracts are measured using a BCAAssay Kit (Pierce, Rockford, Ill.) and 10 μg of each is run on a 10%acrylarnide Tris-glycine gel (Novus Biologicals, Littleton Colo.),transferred to nitrocellulose membranes and probed with a monoclonalantibody to human HIF-1α (BD Transduction Labs, Lexington, Ky.).Compounds for pharmaceutical use are selected based on an 140 kDaltonprotein recognized by the anti-HIF-1α antibody.

Example V

Any compound of the present invention can be screened by measuring theamount of VEGF protein in cardiac tissue after treatment. Sprague Dawleyrats are treated with IV infusions of a test compound for 6 to 12 hr.Animals are euthanized by exsanguinations. Hearts are removed and frozenin liquid N₂. To extract VEGF, pieces of cardiac tissue are homogenizedon ice in 10 mM Tris, 2 mM MgCl2, 150 mM NaCl. 1%

riton X-100 and protease inhibitors (Complete™ Proteinase Inhibitors,Boehringer Manheim). Aliquots of the crude homogenate are sonicated andcentrifuged at 11,000×g for 10 min at 4° C. The supernates are analyzedfor VEGF content using a ELISA kit (R&D Systems). Total proteinconcentration of the crude homogenate is also determiner using a BCAAssay kit (Pierce). Final VEGF levels are expressed as a percentage ofextractable VEGF per mg of protein. Compounds for pharmaceutical use areselected based on highest percentages.

Example VI

Any compound of the present invention can be screened by measuring theamount of EPO in serum. After the compounds are administered toSpraque-Dawley rats, blood is drawn and allowed to clot in polypropylenetubes for 2 hrs at room temperature. Clotted blood is precipitated bycentrifugation and serum supernates are collected and analyzed using anEPO-Trac™ ¹²⁵I Radioimmunoassay Kit (Diasorin, Stillwater, Minn.)according to the instruction protocol provided. Prior to analysis, serumsamples are diluted 1:4 or 1:8 in EPO-Trac standard buffer. Final valuesare corrected to account for the dilution. Compounds for pharmaceuticaluse are selected based on their ability to induce the highest plasma EPOvalues.

It is contemplated that not only are the present examples non-limiting,but also may be used in combination to select a compound of the presentinvention to the desired therapeutic goal(s) such as increasingvascularization of tissue in a subject. To this end, a compound that isfound to induce the VEGF-luciferase reporter of Example I can be testedin the VEGF-ELISA assay of Example II to determine if the compoundstimulates VEGF protein production from an endogenous VEGF gene.Further, the compound can then be tested in the gene selectivity panelassay of Example III to assess the specificity of the response. Furtherstill, the compound can be assessed in the immunoblot assay for HIF-1 ofExample IV to determine if the compound increases HIF-1. Further evenstill, the compound can be further tested in vivo for its ability toincrease VEGF protein expression in rat tissue of Example V or increaseEPO protein levels in rat serum of Example VI.

IX. EXAMPLES Compositions and Methods of Use

The compounds of the invention are useful to prepare compositions forthe treatment of ailments associated with hypoxia. The followingcomposition and method examples do not limit the invention, but provideguidance to the skilled artisan to prepare and use the compounds,compositions and methods of the invention. The skilled practitioner willappreciate that the examples may be varied based on the condition beingtreated and the patient.

Example A

A tablet composition for oral administration, according to the presentinvention, is made comprising: Component Amount (mg per tablet) Compoundof Example 3 5 Microcrystalline Cellulose 100 Sodium Starch Glycollate30 Magnesium Stearate 3

When administered orally once daily, the above composition substantiallyincreases EPO in a subject suffering from anemia.

Example B

A capsule for oral administration, according to the present invention,is made comprising: Component Amount (% w/w) The compound of Example 1415% Polyethylene glycol 85%wherein 1.5 grams of the compound is placed in a standard gelatincapsule.

A human subject suffering from angina is treated by a method of thisinvention. With a regimen of three capsules per day administered orallyto the subject, the patient's angina is relieved. At the end of thetreatment period, the subject is examined and is found to have increasedvascularization to the once ischemic cardiac tissue.

Example C

A topical composition for local administration, according to the presentinvention, is made comprising: Component Composition (% w/v) Thecompound of Example 16 0.20 Benzalkonium chloride 0.02 Thimerosal 0.002d-Sorbitol 5.00 Glycine 0.35 Sensates, including oil 0.075 ofwintergreen Purified water q.s. Total = 100.00

A diabetic subject suffering from a non-healing wound applies thetopical to the wound twice a day. After one month, the wound issubstantially healed.

While particular embodiments of the subject invention have beendescribed, it would be apparent to those skilled in the art that variouschanges and modifications to the compositions disclosed herein can bemade without departing from the spirit and scope of the invention. It isintended to cover, in the appended claims, all such modifications thatare within the scope of this invention.

1-14. (canceled)
 15. A method of increasing vascularization of tissue ina mammalian subject in need of such treatment comprising administeringto said subject a safe and effective amount of a compound having thestructure:

wherein: (a) X is CH; and (b) R6 is selected from the group consistingof nil, methyl, ethyl, methoxy, ethoxy, amino and halo; or an opticalisomer, diastereomer or enantiomer, or pharmaceutically-acceptable salt,or biohydrolyzable amide, ester, or imide thereof.
 16. The method ofclaim 15, wherein the compound is selected from the group consisting of2-(5-Phenyl-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol;2-(1-Pyridin-2-yl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol;2-[5-(4-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol;2-[5-(2-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol;and2-[5-(2-hydroxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.17. The method of claim 16, wherein the compound is selected from thegroup consisting of2-(5-Phenyl-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol.
 18. Themethod of claim 16, wherein the compound is selected from the groupconsisting of2-(1-Pyridin-2-yl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol.
 19. Themethod of claim 16, wherein the compound is selected from the groupconsisting of2-[5-(4-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.20. The method of claim 16, wherein the compound is selected from thegroup consisting of2-[5-(2-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.21. The method of claim 16, wherein the compound is selected from thegroup consisting of2-[5-(2-hydroxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.22. A method of increasing erythropoietin in a mammalian subject in needof such treatment comprising administering to said subject a safe andeffective amount of a compound having the structure:

wherein: (c) X is CH; and (d) R6 is selected from the group consistingof nil, methyl, ethyl, methoxy, ethoxy, amino and halo; or an opticalisomer, diastereomer or enantiomer, or pharmaceutically-acceptable salt,or biohydrolyzable amide, ester, or imide thereof.
 23. The method ofclaim 22, wherein the compound is selected from the group consisting of2-(5-Phenyl-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol;2-(1-Pyridin-2-yl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol;2-[5-(4-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol;2-[5-(2-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol;and2-[5-(2-hydroxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.24. The method of claim 23, wherein the compound is selected from thegroup consisting of2-(5-Phenyl-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol.
 25. Themethod of claim 23, wherein the compound is selected from the groupconsisting of2-(1-Pyridin-2-yl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol.
 26. Themethod of claim 23, wherein the compound is selected from the groupconsisting of2-[5-(4-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.27. The method of claim 23, wherein the compound is selected from thegroup consisting of2-[5-(2-Methoxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.28. The method of claim 23, wherein the compound is selected from thegroup consisting of2-[5-(2-hydroxy-phenyl)-1-pyridin-2-yl-4,5-dihydro-1H-pyrazol-3-yl]-phenol.